Various natural and recombinant proteins have medical and pharmaceutical utility. Once they have been purified, separated, and formulated, they can be parenterally administered to disadvantaged hosts. However, parenterally administered proteins may stimulate an immune response, may be relatively water insoluble, and may have suboptimal pharmokinetic behavior. Consequently, it can be difficult to achieve therapeutically useful blood levels in patients.
These problems may be overcome by conjugating the proteins to polymers. For example, polyethylene glycol (PEG) can be conjugated to proteins for various purposes. Davis et al. U.S. Pat. No. 4,179,337 discloses conjugating polyethylene glycol to polypeptides, such as enzymes and insulin. Davis et al. made these conjugates so that the protein would be less immunogenic and would retain a substantial proportion of its physiological activity. Davis et al. also disclose methods for placing a reactive group on PEG and subsequently conjugating it to a protein. Iwashita et al. U.S. Pat. No. 4,412,989, disclose covalently conjugating polyethylene glycol to an oxygen carrying molecule. This conjugate is useful as a blood substitute. Veronese et al., Applied Biochem. and Biotech., 11: 141-152 (1985) disclose activating polyethylene glycols with phenyl chlorofonnates to modify a ribonuclease and a superoxide dismutase. Katre et al. U.S. Pat. No. 4,766,106 also disclose solubilizing proteins by polymer conjugation. For example, PEG, and other polymers, are conjugated to recombinant proteins to reduce immunogenicity and increase in vivo blood levels, among other things. These compounds may more specifically include interleuldn-2 (IL-2), interferon-.beta. (IFN-.beta.), immunotoxins, and other proteins that share similar characteristics. Nishimura et al., European Patent Application 154,316 and Tomasi Interational Application Number PCT/US85/02572, disclose similar subject matter.
The process for attaching PEG to these useful recombinant proteins is important. Accordingly, the present invention is an advantageous modification in the process for preparing a PEG active ester.